HepG2-derived hepatoblastoma cells (2.2.15) which actively produce hepatitis B virus (HBV) were cultured in the presence or absence of the antiretroviral agents 2',3'-dideoxyguanosine (ddG), 2',3'-dideoxyinosine (ddI), or 3'-azido-2',3'-dideoxythymidine (AZT). All of these agents diminished the viral replication in 2.2.15 cells as assessed by the amount of extrachromosomal HBV DNA without affecting cellular growth. Among the three dideoxynucleosides, ddG was the most potent agent, diminishing viral replication by as much as 97%. Northern blot analysis revealed no apparent difference between treated and untreated cells in the pregenomic RNA profile, suggesting that dideoxynucleosides suppress HBV replication at the stage of reverse transcription. The effect of varying the time of drug exposure showed that these agents can suppress HBV replication even when added late in culture, suggesting that if de novo DNA synthesis mediated by reverse transcriptase is continuously suppressed, the extrachromosomal DNA molecules are unstable and disappear after a certain period of time. HPLC analyses using (3)H-ddG showed that anabolic phosphorylation of ddG to an assumed active metabolite, ddGTP, occurs in 2.2.15 cells. These data suggest that 2',3'-dideoxynucleosides may be potential experimental drugs for the treatment of HBV infection by targeting the reverse transcription step, although the data do not address the in vivo toxicity profile or therapeutic index.